Tobacco smoking is highly prevalent in the HIV-infected population, and is known to exacerbate HIV pathogenesis. HIV infection to the brain cells such as microglia and macrophages are known to cause HIV- associated neurocognitive disorder (HAND) in 50% of the HIV+ population. Since tobacco constituents, mainly benzo(a)pyrene (BaP), also cause neurotoxicity, together they may further exacerbate neurotoxicity in HIV- infected smokers. Extracellular vesicles, especially exosomes (30-100 nm), which are gaining importance as biological markers and carriers for cancer therapies, have been proposed to play a critical role in HIV pathogenesis. Recent studies indicate that exosomes secreted from HIV-infected monocytes integrate with adjacent uninfected monocytes and facilitate HIV infection. However, there is nothing known about the role of exosomes in tobacco-mediated HIV pathogenesis and neurotoxicity. The long-term goal of this proposal is to identify the key components of tobacco/HIV-induced exosomes from macrophage/microglia that are responsible for exacerbated HIV pathogenesis and neurotoxicity. Our objective in this proposal is to identify exosomal factors in monocyte-derived macrophages (MDM) and underlying mechanism that are responsible for tobacco-mediated increased HIV replication and neurotoxicity. The central hypothesis is that exosomal components, especially related to oxidative stress pathway, that are released from MDM upon exposure to tobacco constituents are the key mediator for HIV replication and neurotoxicity. We will test the hypothesis as follows. Specific Aim 1: Determine the contribution, and underlying mechanism, of CSC/BaP and HIV in regulating secretion of exosomes/exosomal AOEs from MDM: Our working hypothesis is that exposure of CSC/BaP and HIV to MDM decreases secretion of exosomes and exosomal AOEs through their decreased synthesis. Specific Aim 2: Determine the contribution and underlying mechanism of exosomes/exosomal AOEs towards HIV replication in MDM and neurotoxicity. Our working hypothesis is that exosomes, which are derived from CSC/BaP-treated MDM, increase HIV replication, and exposure of CSC/BaP and HIV together to neuronal cells increases neurotoxicity. Upon successful completion of the proposed research, we will have established that exosomal AOEs derived from CSC/BaP-treated MDM are critical for HIV pathogenesis and neurotoxicity. Such outcome will open a new avenue in understanding the mechanisms of smoking-mediated HIV pathogenesis and neurotoxicity. The knowledge obtained will provide an incentive to evaluate exosomes as biological markers and/or novel carriers for therapies in HIV-infected smokers.